Photodynamic therapy (PDT) treated tumor cells have proven to be efficacious anti-tumor vaccines in both therapeutic and preventative settings in murine models of cancer. However the mechanism by which PDT enhances tumor immunogenicity is unclear. PDT treatment of tumor cells has been shown to induce the release of "danger" signals capable to stimulating anti-tumor immunity. Danger signals are recognized by danger signal receptors, Toll-like receptors (TLRs) and NOD-lie receptors (NLRs). We and others have shown that PDT treated tumor cells contain TLR ligands. In a novel finding we now show that PDT-treated tumor cells stimulate the NLR family of danger receptors. We hypothesize that activation of TLR and NLR is critical to the enhancement of anti-tumor immunity by PDT. Furthermore we hypothesize the combination of PDT or PDT vaccines with therapies that augment induction of danger signal pathways will further enhance the induction of anti-tumor immunity and will lead to enhanced control of distant disease in more aggressive, spontaneously metastasizing tumor models. Therefore we propose to combine PDT with agents that enhance the immune response through signaling pathways complimentary to those induced by PDT-treated tumor cells, in order to augment the anti-tumor immune response induced by PDT vaccines or PDT. Finally we have shown that PDT-treated tumor cells can be used in an adjuvant setting to enhance anti-tumor immunity following surgical resection in a pre-clinical model of melanoma. We now propose to extend these findings to a clinical setting by investigating the safety and immunogenicity of an autologous PDT vaccine for advanced stage III in transit melanoma following surgical resection of tumors. The overall goal of this renewal is to understand the mechanisms by which PDT enhances tumor cell immunogenicity with the long-term goal of developing clinical PDT protocols that enhance anti-tumor immunity and combat secondary disease. Our current objective is to understanding the mechanisms by which PDT promotes anti-tumor immunity in order to facilitate the development of clinical PDT regimens that both control long-term tumor growth and promote anti-tumor immunity. Three specific aims are planned to enable us to test our hypotheses and achieve this objective. The first two aims are pre-clinical and synergistic;Specific Aim 1 examines the mechanism by which PDT-treated tumor cells activate the NLR family member, NALP3. Specific Aim 2 explores the use of TLR agonists to augment the anti-tumor effects of PDT-generated vaccines. The application culminates in Specific Aim 3, which tests the clinical application of PDT-generated vaccines. PDT is an effective therapy for a growing number of malignancies, however optimization of PDT has been hindered by the complexity of the therapy. Our studies will both aid in the optimization of PDT and permit exploitation of its ability to enhance anti-tumor immunity. PUBLIC HEALTH RELEVANCE: Patients with advanced lymphatic metastatic melanoma have a poor long-term prognosis. The primary treatment for melanoma remains surgical excision, which can be curative for early disease, but has minimal effects on metastatic disease. The overall prognosis for patients with melanoma has not improved over the last 30 years despite substantial effort. In recent years a number of melanoma vaccines have been studied;however in spite of promising Phase I-II studies, no durable responses have been observed in Phase III randomized trials. These disappointing results are likely a result of a number of factors including tumor- induced immune suppression and tolerance and continued tumor growth. Thus efforts have focused on the development of vaccines that augment anti-tumor immunity by overcoming the natural suppressive factors, in an adjuvant setting, such as in combination with surgery. Our pre-clinical studies suggest that PDT directly alters tumor cell immunogenicity, in part through induction of "danger" signals. Our proposal is aimed at 1) understanding the response of immune cells to danger signals generated by PDT treatment of tumor cells and 2) enhancement of anti-tumor immunity by PDT vaccines through the use of combination treatments. We will use the results of these studies as guidelines for a Phase I trial that investigates the use of PDT-generated vaccines as adjuvants to surgical removal of tumors in patients with Stage III in-transient melanoma.